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Transgenic Xenopus line expressing the sleeping beauty transposase

Our approach for mobilizing gene trap transposons in vivo was to generate two types of transgenic lines; one expressing the SB transposase specificically in the male germline, and the other set of lines contain the gene trap transposons in the genome of the animals.

These two lines are crossed together and, in the male progeny that contain both sets of transgenes, the gene trap transposon is mobilized in the male germline and these "hops" are revealed in the next generation (i.e. his progeny). Thus double transgenic males are crossed to wild type females and the progeny are screened for novel gene trap insertions. In order to easily identify which animals contain each transgene, we generated each set of transgenic lines linked to different eye colour markers.

Specifically, we generated the transgenic lines expressing the transposase linked to a transgene containing the gamma-crystallin promoter driving green fluorescent protein (GFP). These transgenic lines are easily identified by having green eyes. The lines that contain the gene trap transposons are linked to the gamma-crystallin promoter driving red fluorescent protein (RFP). These transgenic lines are easily identified by having red eyes. When the two sets of lines are crossed with each other, those that are transgenic for both transgenes will express a combination of green and red in their eyes, which is detected as yellow eyes.

For the lines that we generated which express SB transposase in the male germline, we used two different promoters. One of the promoters we used was the mouse protamine promoter. The other promoter we used was the Xenopus SP4 promoter, which we cloned using genomic PCR. Using RT-PCR, we confirmed that both transgenic lines express Sleeping Beauty in the testes of males. These lines are of great benefit for any group who is interested in using Sleeping Beauty transposition to mobilize transposons in the male germline of Xenopus tropicalis.